Lactic acid bacteria, which are the most important microorganisms among food microorganisms, have acquired the GRAS (generally recognized as safe) status, and thus have been used in various foods. These lactic acid bacteria have plasmids, bacteriophages, transposons and the like, thus making it possible to develop vectors for introducing genes therein. Also, these lactic acid bacteria are easily transformed according to conventional methods known in the art, and are considered to be most suitable for edible purposes, because edible selectable marker genes have been established. In addition, lactic acid bacteria have the effects of inhibiting harmful intestinal bacteria, cleaning intestines, lowering blood cholesterol levels, increasing the nutritional value, inhibiting infection with pathogens and alleviating liver cirrhosis, as well as an anticancer effect and the effect of boosting the immune system through macrophage activation.
In order to produce useful heterologous proteins in lactic acid bacteria, highly efficient promoters (van der Vossen et al., Appl. Environ. Microbiol., 53:2452, 1987; Koivula et al., Appl. Environ. Microbiol., 57:333, 1991; Pascalle et al., Appl. Environ. Microbiol., 62:3662, 1996) are required, but studies on the genomes of lactic acid bacteria are still very insufficient. Among the genomes of lactic acid bacteria, only constitutive the genomes of Bifidobacterium longum NCC 2705, Enterococcus faecalis V583 and Lactobacillus plantarum WFCS 1 have been sequenced to date, and studies on the genome sequencing of a variety of lactic acid bacteria are currently in progress. In addition, as promoters isolated from lactic acid bacteria, only promoters derived from the genomes of Streptococcus thermophilus A054, Lactococcus lactis MG1614 and Lactococcus cremoris Wg2 (Slos et al., Appl. Environ. Microbiol., 57:1333,1991; Koivula et al., Appl. Environ. Microbiol., 57:333, 1991; van der Vossen et al., Appl. Environ. Microbiol., 53:2452, 1987)_are known to date.
Recently, in USA and Europe, studies on the development of live vaccines using lactic acid bacteria, studies on vehicles for delivering useful hormone drugs into the intestines, and studies on the establishment of efficient genetic resources therefor and the development of insertion vectors for lactic acid bacteria, are being conducted. Particularly, the utility of lactic acid bacteria as vaccine vehicles has been highly evaluated, because unmethylated CpG DNA, lipoteichoic acid, peptidoglycan and the like, which are contained in lactic acid bacteria in large amounts, are known to function as adjuvants. In addition, lactic acid bacteria have many advantages in that they can induce intestinal mucosal immunity, because they show resistance to bile acid and gastric acid to make it possible to deliver antigens to the intestines (Seegers, Trends Biotechnol., 20:508, 2002).
However, in order for lactic acid bacteria to be used as vaccine vehicles, it is required to develop a technology of presenting antigen proteins for the production of disease-preventing antibodies to the inside or outside of bacterial cells so as to allow antigen-antibody reactions to occur smoothly. In fact, various study results, which indicate that lactic acid bacteria are suitable as vaccine vehicles, have been published. Examples of these studies include the examination of the antibody-inducing capacity of lactic acid bacteria, in which the L1 protein of human papilloma virus (HPV) is expressed (Aires et al., Appl. Environ. Microbiol., 72:745, 2006), and the examination of the disease-treating effects of a lactic acid bacterial strain which secrets and expresses IL-2 (interleukin-2) (Steidler et al., Nat. Biotechnol., 21:785, 2003).
As described above, the development of various applications of lactic acid bacteria expressing target proteins, and scientific studies on the lactic acid bacteria, have been actively conducted, there are problems in that the expression levels of the target proteins are insufficient and that, when inducible promoters are used, the continued expression of the target proteins in vivo can also be impossible.
Previously, the present inventors developed a novel vector for effectively expressing exogenous protein on the surface of microorganisms, using a poly-gamma-glutamic acid synthetase complex A (pgsBCA) gene, derived from Bacillus subtilis var. Chungkookjang, as a novel surface anchoring motif, and a method for expressing a large amount of target proteins on the surface of microorganisms transformed with said vector (Korean Patent Registration No. 10-0469800).
Accordingly, the present inventors have understood that, if a vector capable of stably expressing an antigen or an antigen determinant at a high level in lactic acid bacteria using the surface anchoring motif described in said patent is developed, it will be possible to develop a vaccine, which is compatible with the human body and can efficiently induce an immune response, because the antigen is exposed on the surface of lactic acid bacteria. Based on this understanding, the present inventors have conducted a process of screening a promoter capable of highly expressing a target protein in lactic acid bacteria and, as a result, have found that, when an aldolase gene promoter is used, the expression of target genes is increased by at least 2.5 times compared to when a conventional promoter is used, thereby completing the present invention.